crj /t r(331Gi~-------i loo~13161 I I a IJI .a Y (1 a 12!,2Egn vo o r t; d t O c a I~ri a: P: o \"1 I~ W (6 O ~,5 3 c3 O O ~dX ~Jo NTOZO 43 C 0 2· a wJ I- w 9 in" Y c, f o: I,S 3a ;Q 2 ~ec 4 ~t zX s~ IJ ·n a a ·5 h O a LLC L3 t '~ z a g 2 Y ul o83w v 3'3 o Ha a O Q 03 I,a: -Ir 3wh JI ~· r , I bE 4 Z: a J r O wI S Z 0 J bP1 J r a 5 " uO 2 rw Ow S 0 a .O'o~'C'~w~"7)a2 i, ~" O ~r W P C;u JcBO CO r 3 Zi3 2 a: ~ C C]0: JZ 3~Cd o J cl z f o: do: t w JJ 2 ~ gwn 2 3 N c~oO gP z ~ .o IJB x vj ~~ 1- ale C~O; f d Q Prr ·n o,c~l~e Z·o w (1 Q> 0 o~l-d~E P w 4 W V1 -Y Y ~t 3 es~ ~ E N 2 o. 4 C1 ~3 d W w d O ct~J ·.r O Cw c 0 Ow6 P P1 (alZw WOdQr~ ~i s "z~r: · ·In s SCd ·.I O Zj2 Z' d dj t O 4: Jld o o· Q IJ Z r· C]")d JwnO ao t d ·. i o. Z gxo aP: IJ 5 3 c) W LO I J IZ~ "aa " lid C'rl '·1 0' 2 m~Lt : · r.·! O ·; o w BA~Co document for legal Services : Health Canada 19 May 1999 ~~~ PRA~N N;t~Dgpn To CkU. k '/ r! I \ BypaSs Scmpk Sampk In .LN riltrqgq II II ~To CoLL. \,* 5,16 S~pk~ Itn. V~nt BATCo document far Legal Services : Health Canada 19 May 1999 I \ I I \I/ 1C I ct s o w P O U BATCo document for Legal Services : Health Canada 19 May 1999 mi Z be do 1J Z 4 II ill I Q W Z 3 O E 3 a d o II g d W Z o Eti LL BATCo document for Legal Services : Health Canada 19 May 1999 ___~__·-t· C· INDEX FILE NOTE 1055 ~aln~l~oac · 31st January, 19BO res DETERMINATION a ClTRATES D PAPER The automated method for the determination of citrate in cigarette paper as detailed in B ~ ~a Report No, 66-26B, author J, T, Lead, has been examined, Two changes have been made, one In the interest of laboratoV safety lad the other due to changes in availability of materials. The changes made are: i, The Maste reaction mixture is diluted with an eqnal f!at of water and mixed, prior to collection in a waste bottle. This eliPinates the possibilit! of heat build up if water is aCded to the waste solvent bottle when the analyzer is p~ped out at the beginning or end of a run, 2, The 'abbr latex' pumT3 tube has been replaced with silicone pump tube which is now a standard supply its. it is essential for the spsta to be sat up under a fume hood or in a f~e clpboard, me possibility of determining citrate on the acid cxt:act used for phosphate dctenioations has been investigated and found to be impracticable. The presence of acid reduces the sensitiVitlr of the method, Cross checks between the manual and automated rc~ods gh~ good agreement (particularly since the specification for papers is mean 1 0,21 citric aeidJ, BATCo document for legal Services : Health Canada 19 May 1999 TABLE I SAMPLE $ CITRIC ACID MANUAL MFTHOD AUTOANFLYZER METHOD A ,42 ,44 ,41 ,411 ,42 ,41 ,42 .44 ,41 ,46 ,44 .43 AV,,42 AV. ,44 S.D.,011 S.D, ,010 C .92 ,92 ,95 .93 .85 .93 ,93 ,95 ,93 .95 .93 ,5 ,92 Av, ,94 S,D..035 s,D. 1013 BA~Co document for Legal Services : Health Canada 19 May 1999 -3- TABLE TI SYLE r CITRIC ACrD E"JLE~& METHOD Imo~mPxgR METBOD 763 1.11 1.12 .01 765 1,05 1.01 -,04 737 0.98 -,03 141 0,97 0.99 .02 770 1,05 1.04 -,01 772 0,90 0,90 0 ~1 1.56 1.58 .02 ~2 1.56 1.62 .06 834 0.86 0.84 -.02 835 0.88 0.87 1,01 836 0,99 0,96 -,03 837 0,90 0,90 O 838 0,92 0.90 ·,02 839 0,99 0.98 ·.ol 840 0,98 a,98 ' ~O 1.049 1,045 Table I shows repeat analysese of two samPles, Table II shows single results fro fifteen different sqlcg. The revised ncthaC has been written as a draft in GR i DC ~onat and II attached, ip W, J. ROLES, Distribution: B, 8, jaeo Q Pile 10BC A, G, Brown W B, F. Dpond ~ L BATCo document for legal Services : Health Canada 19 Yly 1999 DETEiP1INATION OF CITRATE LEVELS IN CIGARETTE FARER USING A CONTINUOUS fLOW ANALYZER SUKMARY The paper is extracted with water, and citrate in the resulting extract determined colourimctrically after reaction with acetic anhydride and pyridine, using a continuous flow analyser, REAGENTS Acetic a~hvdride, (Cfl,. CO),O, (A.R.) Pyridine, Cs HSh', (A,R~) Tri-sodi~m citrate, dihydrate, Na, Cs H507 2s,a, (A,9.) Colour RE~dent'#iX equal volumes of pyridine and acetic anhy~rii~e in a dry flask just prior to use Standard Solutions Primary - Disolve 1.000g of tri-sodium citrate dihydrate (dried over silica gel for at least 3 hours) in de-ionised water and dilute to 500me in a volumetric flask, Store in a re:ridcerator. Secondary - Transfer 51 10, 15, 20 and~25ml aliqvots of the prixlzr~ standard into 100 me volumetric flasks. Dilute to volume with de-ionised water. These standards are ecoivalent to 0.5, 1.0, 1,5, 2.0 and Z,0%hvdrateC sodium citrate in cigarette paper using the procedure detailed overlel. BATCD document for Legal Services : Health Canada 19 May 1999 -2- APPARATUS 250 me conical flasks with stoppers, A continuous flow analyzes comprising the following modules : i, Sampler, set to run at 60 tests per hour with a sample-to-wash ratio of 2:1, 2, Proportioning pump carrying the following manifold: COLOUR CODE CONTENTS TYPE Purple/white Acetic anhydride/ Silicone pyridine Purple/white Plow cell return Silicons ~Prple/white wz:er Standard Orange/orange Air Standard alack~lack Sample Standard a!uejblue Water wash Standard 3, Colouli~eter with 15mm flow cell, 420nm filters and silicone rubber/P,T.F.E., tubing connections to the flow ce!l~ 4, Recorder, PROCEDURE i. Weigh out accurately, to the~nearcst 0,01g, Ig of chopped up paper Into a 250 me conical flask. 2, Add 50 mi ~e-ionise~ water. 3, StoPper the flask and shake, leave:or 20 minutes shakin~ the flask at intervals. 4. load the sampler plate as follows: i. Duplicate cups of the five standards. 2. Duplicate cups of de-ionised water. 3. A maxirnun~ of twelve r;ample cups and one c3ntrol cup, BATCo document for Legal Senrices : Health Canada 19 May 1999 13- 4, Duplicate cups of de-ionised water, 5. As from 3) the run ending with duplicate standards, 6, Run de-ionised water through the manifold for at least 30 minutes, i. Remove recorder chart and prepare a standard curve by plotting O citrate against peak height, Read off from the standard curve the a citrate in the samples , CA~CIT~ATIONS Calculate the anhyarous citric acid content of the samples 25 follows: e arhydrous citric acid e C x 0,65 where C = 1 of citrate obtained, NOTES i. it is essential to use glass or silicdne tubing where there is contact with the anhydride reagent. REPER~CE "Automateci Determination of Citrate in Cigarette Paper" Brown & Williamsoc, Reasearc~ and Development Department Reoort 66-26, WJ~INMM 4 31st januar!, 1980. ~ BATCo document for Legal Services : Health Canada 19 May 1999 i9i iS ii I tp~ L " J E ·! 1 t j E2 u 1g 3 It P~ P 1 1 I 6 e 1 ·1 f-~ o oi I lI -~ J F~ t I b 4 D r i a'~ v3 h O b 0\ O CD VI BATCo document for Legal Services : Health Canada 19 May 1999 PM~ ICIDS rur~ish tobaccos contain particularly hith concentrations of Ilctbtlbnc~ic, isolole:ic ad I~c~hllrllc!ic acids, These acids may be connected dth Turkish flavour, In smoke, Virginia tobacco delivers highs levels of volatile acids than Burlcg or I~ld ~~B~ Turkish smoke is similar to Virginia smoke uith the imPortant IIEltiOPS that ilB11CliC and B-aethylvaleric acid concentrations are extremely high. alanine is the most predominant free dno acid in smoke, followed by prolioe and glycins. ~SEIIC C~O~S Tbc burning cone of a cigarette padlcas a reducing atDospbcse (typical hidrog~ cdncealration: 82 by volume), Since tobacco con~ains arsenic, Irr~e (ls83), a volatile arsenic compounC which would transfer to smoke, may be formed, a lack of interest in arsenic $s been attributed to a drastic decrease ic the level of arsenic in tobacco in the last few decades, a typical modern value for the level of arsenic in smoke is 0.012 ~glcig~rotro, and a tobacco value is 0,36,7 p.p,m, Older values for tobacco were 13,9 p.p,o. (1933) and 6,1 p,p,m, (1959) but arsenic contlhing insecticides such as lead arsenttcs and paris green (copper Icctate ncta-arscapce) have been phased out, Only ~51 of tobacco arsenic is recovered from rinrtcs smoke; most remains in the ashes and in E~e butt, BATCo document for Legal Services: Health Canada lB May l919 E~C~ eWPOOIS Several investigators have found nickel in mainstream ~~LI but it is felt that this is unlikely to be nickel carbonyl (Bi(~)~), a volatile and dangerous nickel compound, The concentration of nickel in tobacco has been reported to be 2-6 p,p,m,; 2 p~p.m, of nickel coupled dth 2-72 of carbon larxide should yield 1 toxic dose of Ni(M)4, buc the ploduc~ deconpascs at 2000L I typical modern value for the level of nickel in snoke is 0.080 vBlcigarltte, UbOLS The major ·bh lacroPd is y-5utyrolactone, ~ob~cco also contains hgdroxyco~atiPs (~ich contain the lactons grouping), 0f these, scopoletia (hydroIy pCthda comlrin) has been detected in smoke. Angelica lactones may fin in smoke from levulinic acid, I number of very complex laetoner (levantcoolidcd, noImbrtinolide ~PC aigdralctinodulide) be been detected. Iflatbxins are carcinogenic lactones, but these have no: been found in tobacco. They are metabolites of the fungus Asper~llls nlma, thus tobacco fungi cty produce similar mcubolites, though not necessarily lactones. P13 ~IC~S Concentrations are greater in fresh smoke than in aged smoke, and results suggest a mixture of unstable and stable radicals. Stable radical concentrations are highest in virginia smoke and lovelt in Burley smoke, ~xin~ conce~t;ati~s are generated It 600DC, ~Et radicals are thought to be ;iolyaromatic hydrocarbon adic~lr, Concentrations of free radicals are ten times higher in mainstream smoke than sidettrca snotre. Ilthough radicals may interact ith proteins, mainstream and 8idcstreP sorer have Iqual tumour responses on mouse skin. B~Co document for legal Senices : Health Canada 19 May 1999 K1I)ROCEy SU'1PHfDE Deliveries are ranked Virginia) Burley, Turkish, An increase in smoke oh can influence the level of hydrogen sulphide in vapour phase; an 802 reduction can result sin alkaline smoke. Acelare and paper filters have no effect on hydrogen sillphide deliveries, as would be expected for a volatile gas, bur granular carbons efficien:lp remove this constituent frwn smoke, hydrogen sulphide is believed to result from sulphur·fontaining amino acids and proteins, Very Little methionine and cgstine is Present in tobacco, but these acids may be important tobacco protein eonstituenrs, The lowest sulphur levels are found in Virginia tobacco and the highesl· levels in Bryrand tobacco, ORCIIUIIC SIfanES The most significant nitriles are acetonitrile, acrylonitrile and propionieri!e, and a method has been developed in Group ii, a D. Centre for the measurement of these three constituents of smoke.3-cyanopyridi~e (from the pyrolysis pi nicotine) is also important. increased nitrate content of tobacco results in higher acetonitrile and acr~loai:rile deliveries, Glycine also increases acrylbnitrire deliveries. AcerPni:rile delivery increases when tobacco is treated with glucose or sucrose; this suggests that reactions between sugars and nitrogen-containing compounds in sPoke occur, Naphthonltriles and benzonitriles result from the pyrolysis of lysine [high in Surlcy), leucine, t~yptophan thigh in stem), phenylalanine and Paleic hvdrazide, Ihe ~yro!ysis of ~yridine, picolines and methylpyratine also results in the formation of significant amounts of volatile nitriles, BATCo document for legal Services : Health Canada 19 May 1999 SULPHUR DIOTIlDT Sulphur dioxide is a very minor constituent of cigarette smoke, and the concentrations in Virginia.and Burley smokes are very similar. Carbon containing filters remove approximately 501 of the small concentration present, BATCo document for Legal Senices : Health Canada 19 May 1999